A. Angiogenesis and Neovascular Ocular Diseases
A1. Angiogenesis and Angiogenesis-Related Diseases
Angiogenesis is the generation of new blood vessels in a tissue or organ (Carmeliet, 2005, Nature, 438: 932-936). Under normal physiological conditions, humans and animals undergo angiogenesis only in very specific and restricted situations. For example, angiogenesis is normally observed in wound healing, fetal and embryonic development, and formation of the corpus luteum, endometrium and placenta.
Angiogenesis is controlled through a highly regulated system of angiogenic stimulators and inhibitors (Yancopoulos et al., 2000, Nature, 407: 242-248). Angiogenesis is turned on by specific angiogenic molecules such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), angiogenin, transforming growth factor, tumor necrosis factor-alpha (TNF-alpha) and platelet derived growth factor (PDGF). On the other hand, angiogenesis can be suppressed by inhibitory molecules such as interferon-α, thrombospondin-1, angiostatin, and endostatin. It is the balance of these naturally occurring stimulators and inhibitors that controls the normally quiescent capillary vasculature. When this balance is upset, as in certain disease states, capillary endothelial cells are induced to proliferate, migrate and ultimately differentiate.
The control of angiogenesis has been found to be altered in certain disease states and, in many cases, the underlying pathology associated with the diseases is related to uncontrolled angiogenesis. Both controlled and uncontrolled angiogenesis are thought to proceed in a similar manner. Endothelial cells and pericytes, surrounded by a basement membrane, form capillary blood vessels. Angiogenesis begins with the local dissolution of the basement membrane by enzymes released by endothelial cells and leukocytes. Endothelial cells, lining the lumen of blood vessels, then protrude through the basement membrane. Angiogenic stimuli promote endothelial cell migration through the eroded basement membrane. The migrating cells form a “sprout” off the parent blood vessel where the endothelial cells undergo mitosis and proliferate. The endothelial sprouts merge with each other to form capillary loops, creating a new blood vessel.
Persistent, upregulated angiogenesis occurs in many disease states, including tumor growth metastases. The diverse pathological diseases states in which upregulated angiogenesis is present have been grouped together as angiogenic-diseases, angiogenesis-associated or angiogenesis-related diseases.
One example of diseases dependent on angiogenesis is ocular neovascular diseases (Gariano and Gardner, 2005, Nature, 438: 960-966). These diseases are characterized by invasion of new blood vessels into the structure of the eye, such as the retina or cornea. They are the most common cause of blindness and comprise approximately twenty eye diseases. In age-related macular degeneration, the associated visual problems are caused by an ingrowth of choroidal capillaries through defects in Bruch's membrane, with proliferation of fibrovascular tissue beneath the retinal pigment epithelium. Angiogenic damage is also associated with diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, neovascular glaucoma, and retrolental fibroplasias. Other diseases associated with corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, and pterygium keratitis sicca.
Another example of angiogenesis-related disease is rheumatoid arthritis (Bainbridge et al., 2006, Curr Pharm Des, 12: 2631-2644). The blood vessels in the synovial lining of the joints undergo angiogenesis. In addition to forming new vascular networks, the endothelial cells release factors and reactive oxygen species that lead to pannus growth and cartilage destruction. Angiogenesis may also play a role in osteoarthritis. The activation of the chondrocytes by angiogenic-related factors contributes to the destruction of the joint. At a later stage, the angiogenic factors promote new bone growth. Therapeutic intervention that prevents the cartilage destruction could halt the progress of the diseases and provide relief for persons suffering from arthritis.
Chronic inflammation may also involve pathological angiogenesis. Such disease as ulcerative colitis and Crohn's disease show histological changes with the ingrowth of new blood vessels into inflamed tissue. Bartonellosis, a bacterial infection found in South America, in a chronic stage is characterized by proliferation of vascular endothelial cells.
Several lines of direct evidence now suggest that angiogenesis is essential for the growth and persistence of solid tumors and their metastases. To stimulate angiogenesis, tumors upregulate the production of a variety of angiogenic factors, including the bFGF and VEGF (Yancopoulos et al., 2000, Nature, 407: 242-248). However, many malignant tumors also generate inhibitors of angiogenesis, including angiostatin, endostastin, and thrombospondin (Nyberg et al., 2005, Cancer Res, 65: 3967-3979). It is postulated that the angiogenic phenotype is the result of a net balance between these positive and negative regulators of neovascularization. Several other endogenous inhibitors of angiogenesis have been identified, although not all are associated with the presence of a tumor. These include, platelet factor 4, interferon-alpha, interferon-inducible protein 10, which is induced by interleukin-12 and/or interferon-gamma, the 16 kDa N-terminal fragment of prolactin, tumstatin, arresten, canesten, anastellin, vasostatin, and vasohibin.
A2. Ocular Neovascularization and Related Disease States
Pathologic or aberrant angiogenesis/neovascularization, aberrant remodeling, fibrosis and scarring and inflammation occur in association with retinal and ocular ischemic diseases such as age-related macular degeneration (AMD), diabetic retinopathy (DR) and in retinopathy of prematurity (ROP) and other developmental disorders (Eichler et al., 2006, Curr Pharm Des, 12: 2645-60) as well as being a result of infections and mechanical or chemical injury to the cornea and the eye in general (Ciulla et al., 2001, Curr Opin Opthalmol, 12: 442-9; Dart et al., 2003, Eye, 17: 886-92).
Diabetic retinopathy is a leading cause of blindness in adults of working age. The leading cause of vision loss for Americans under the age of 65 is diabetes; 16 million individuals in the United States are diabetic and 40,000 per year suffer from ocular complications of the disease, often a result of retinal neovascularization. DR, therefore, is a retinal microvascular disease that is manifested as a cascade of stages with increasing levels of severity and a worsening prognosis for vision. DR is broadly classified into 2 major clinical stages: nonproliferative diabetic retinopathy and proliferative diabetic retinopathy (PDR), where the term “proliferative” refers to the presence of preretinal neovascularization (PNV) emanating from the retina into the vitreous. Ocular neovascularization occurs in areas where capillary occlusions have developed, creating areas of ischemic retina and acting as a stimulus for neovascular proliferation that originate from pre-existing retinal venules at the optic disk and/or elsewhere in the retina posterior to the equator of the eye. Vitreous haemorrhage and tractional retinal detachment from PDR can cause severe vision loss (Boulton et al., 1997, Br J Ophthalmol, 81: 228-223). Diabetic macular edema (DME) is a further common cause of blindness (Levin, 2001, J Glaucoma 10:19-21; Stefansson et al., 1992, Am J. Ophthalmol. 113:36-38). A clinical hallmark of PDR includes the increased vascular permeability, leading to DME, and endothelial cell proliferation.
Age-related macular degeneration is a leading cause of vision loss in people over 65 years old. For example, AMD affects 12-15 million American over the age of 65 and causes vision loss in 10-15% of them. In contrast to ROP and PDR, in which neovascularization emanates from the retinal vasculature and extends into the vitreous cavity, AMD is associated with neovascularization originating from the choroidal vasculature and extending into the subretinal space. Choroidal neovascularization (CNV) causes severe vision loss in AMD patients because it occurs in the macula, the area of retina responsible for central vision (Kitaoka et al., 1997, Curr Eye Res, 16:396-399).
Multiple theories exist but, the exact etiology and pathogenesis of AMD are still not well understood. Aging is associated with cumulative oxidative injury, thickening of Bruch's membrane and drusen formation. Oxidative stress results in injury to retinal pigment epithelial cells (RPE) and, in some cases, the choriocapillaris (Zarbin, 2004, Arch Opthalmol, 122: 598-614; Gorin et al., 1999, Mol V is, 5: 29). Injury to RPE likely elicits a chronic inflammatory response within Bruchs membrane and the choroid (Johnson et al., 2000, Exp Eye Res, 70: 441-9). This injury and inflammation fosters and potentiates retinal damage by stimulating CNV and atrophy (Zarbin, 2004, Arch Opthalmol, 122: 598-614; Witmer et al., 2003, Prog Retin Eye Res, 22: 1-29). CNV results in defective and leaky blood vessels (BV) that are likely to be recognized as a wound (Kent and Sheridan, 2003, Mol V is, 9: 747-55).
Retinopathy of prematurity (ROP) occurs most prominently in premature neonates. In various cases, the retina becomes completely vascularized at full term/near birth. In the premature baby, the retina remains incompletely vascularized at the time of birth. Rather than continuing in a normal fashion, vasculogenesis in the premature neonatal retina becomes disrupted. Abnormal new proliferating vessels develop at the juncture of vascularized and avascular retina. These abnormal new vessels grow from the retina into the vitreous, resulting in haemorrhage and tractional detachment of the retina (Neely et al., 1998, Am. J. Pathol, 153:665-670). It is estimated that visual impairment from this disease affects 3400 infants and causes blindness in 650 infants annually in the United States. Angiogenesis is the hallmark of this debilitating condition.
Others retinal diseases associated with retinal neovascularization include sickle cell retinopathy, retinal vein occlusion, certain inflammatory diseases of the eye, ocular tumorigenesis, Eale's disease, myopic choriodal neovascularization, and polypoidal choriodal vasculopathy. These, however, account for a much smaller proportion of visual loss caused by ocular neovascularization (Neely et al., 1998, American J. of Path. 153:665-670).
Corneal neovascularization, the abnormal formation of blood vessels in the cornea, is a common and serious complication of many corneal diseases and is a major cause of blindness that affects millions of people (Adamis, 2005, Retina, 25: 111-118). The condition is associated with severe visual impairment and is a high risk factor for graft rejection after allograft corneal transplantation. In addition, corneal neovascularization and subsequent opacification remain the most frequent causes of blindness after severe alkali burn trauma. To date, there are no pharmacological or surgical treatment options for the inhibition of corneal neovascularization that have been proven to be both safe and effective. Despite the routine use of topical steroids, the inflammatory response can lead to oedema, lipid deposition and corneal scarring that may not only significantly alter visual acuity, but also worsen the prognosis of subsequent penetrating keratoplasty. In addition, longer-term use of these drugs can lead to various adverse side effects such as cataracts, glaucoma, infection, and delay corneal epithelial healing.
VEGF plays a dominant role in iris neovascularization and neovascular retinopathies. While reports clearly show a correlation between intraocular VEGF levels and ischemic retinopathic ocular neovascularization, FGF likely plays an essential role. Basic FGF is known to be present in the normal adult retina, even though detectable levels are not consistently correlated with neovascularization. This may be largely due to the fact that FGF binds very tightly to charged components of the extracellular matrix and may not be readily available in a freely diffusible form that would be detected by standard assays of intraocular fluids. Furthermore, overexpression of bFGF in the eye does not stimulate neovascularization because it is sequestered (Osaki et al., 1998, Am J Pathol, 153: 757-765), but bFGF does contribute to choriodal neovascularization when there is tissue disruption from the disease process itself or attempts at treatment (Yamada et al., 2000, J Cell Physiol, 185:135-142).
Viable and approved current treatments for diseases related to ocular neovascularization are limited. The approved treatments for AMD are photodynamic therapy with VISUDYNE® (QLT/Novartis) and intravitreal injection of Macugen® (pegaptanib) (Eyetech/Pfizer) or Lucentis® (ranibizumab) (Genentech). Laser photocoagulation alone or photodynamic therapy with VISUDYNE® are therapies that involve laser-induced occlusion of the affected vasculature, which can result in localized damage to the retina. Macugen® (Eyetech/Pfizer) is an anti-VEGF aptamer that binds to VEGF165 preventing ligand-receptor interaction and is labeled for intravitreal injections every 4 weeks. Lucentis® (Genentech) is a humanized anti-VEGF antibody fragment that also binds directly to all isoforms of human VEGF and is labeled for intravitreal injections every 6 weeks. A variety of other pharmacologic therapies are undergoing clinical evaluation for AMD, such as RETAANE® 15 mg (anecortave acetate suspension, Alcon Research, Ltd.), Envision (squalamine, Genera), the VEGF R1R2 Trap, (Regeneron), Cand5 (anti-VEGF siRNA, Acuity), Sirna-027 (anti-VEGFR1 siRNA, SIRNA/Allergan), a topical receptor tyrosine kinase antagonist (TargeGen), sirolimus (rapamycin, MacuSight), etc.
Grid and pan retinal laser photocoagulation are the only proven options currently available for patients with diabetic macular edema or PDR, respectively. Multifocal laser photocoagulation may reduce retinal ischemia and inhibit angiogenesis by destroying healthy tissue and thus decreasing the total metabolic demand of the retina. Laser photocoagulation may also modulate the expression and production of various cytokines and trophic factors. Unfortunately, laser photocoagulation is a cytodestructive procedure and the visual field of the treated eye is irreversibly compromised. Surgical interventions, such as vitrectomy and removal of preretinal membranes, are widely used with or without laser treatment. Similar to the AMD trials, various pharmacologic agents are in clinical trials for DME, such as ARXXANT™ (ruboxystaurin mesylate, Lilly), RETISERT™ (fluocinolone acetonide, Bausch & Lomb), Posurdex (fluocinolone acetonide erodible implant, Occulex/Allergan), I-vation (nonerodible Dexamethasone implant, Occulex), Medidur (fluocinolone acetonide erodible implant, Alimera), etc. Intravitreal or periocular injection of triamcinolone acetonide, a corticosteroid (Kenalog®, Schering-Plough), and intravitreal Avastin® (anti-VEGF Mab (bevacizumab), Genentech) are also being used “off-label” for the treatment of both macular edema and wet AMD.
Anti-VEGF therapies represent a recent, significant advance in the treatment of exudative AMD. However, the phase III VISION Trial with PEGAPTANIB, a high affinity aptamer which selectively inhibits the 165 isoform of VEGF-A, demonstrated that the average patient continues to lose vision and only a small percent gained vision (Gragoudas et al., 2004, N Engl J Med, 351: 2805-16). Inhibition of all isoforms of VEGF-A (pan-VEGF inhibition) with the antibody fragment RANIBIZUMAB yielded much more impressive results (Brown et al., 2006, N Eng J Med, 355:1432-44; Rosenfeld et al., 2006, N Eng J Med 355:1419-31). The 2 year MARINA trial and the 1 year ANCHOR trial demonstrated that approximately 40% of patients achieve some visual gain. Although these results represent a major advance in our ability to treat exudative AMD, they also demonstrate that 60% of patients do not have visual improvement. Furthermore, these patients had to meet strictly defined inclusion and exclusion criteria. The results in a larger patient population may be less robust. In addition, adverse effects on neurons and vessels have been observed in primates after a single administration of the humanized anti-VEGF antibody (Bevacizumab) (Peters et al., 2007, Am J Ophthalmol, 91: 827-831) and sporadic case reports of complications of anti-VEGF therapy related to regression of blood vessels, increased risk for stroke and myocardial infarction, and local side effects due to the intravitreal application mode have appeared (Fraunfelder et al., 2005, Drugs Today, 41: 703-709; Tobin et al., 2006, Insight, 31: 11-14; Rosenfeld et al., 2006, Ophthalmol Clin NA, 19: 361-372; Baffert et al., 2006, Am J Physiol Heart Circ Physiol, 290: H547-H559; Hurwitz et al., 2004, Clin Colorectal Cancer, 4(suppl): 2: S62-S68). The limited efficacy and potential adverse effects of currently implemented therapies emphasize the need for alternative therapeutic strategies.
B. Ischemia-Reperfusion Injury and Myocardial-Related Disease States
Ischemia-reperfusion injury (I/R injury) refers to an event in which the blood supply to a tissue is obstructed, such as myocardial infarction. Whenever there is a transient decrease or interruption of blood flow the net injury is the sum of two components: the “direct” injury occurring during the ischemic interval and the “indirect” or reperfusion injury which follows. Reperfusion injury can be defined as the damage that occurs to an organ that is caused by the resumption of blood flow after an episode of ischemia. This damage is distinct from the injury resulting from the ischemia per se. One hallmark of reperfusion injury is that it may be attenuated by interventions initiated before or during the reperfusion. Reperfusion injury results from several complex and interdependent mechanisms that involve the production of reactive oxygen species, endothelial cell dysfunction, microvascular injury, alterations in intracellular Ca2+ handling, changes in tissue metabolism, and activation of neutrophils, platelets, cytokines and the complement system. All of the deleterious consequences associated with reperfusion constitute a spectrum of reperfusion-associated pathologies that are collectively called reperfusion injury. Reperfusion injury can extend not only acutely, but also over several days following the tissue attack.
During blood vessel obstruction, the endothelial tissue lining the affected blood vessels becomes “sticky” and begins to attract circulating white blood cells (Tohoku, 2008, J Exp Med, 215: 257-266). The white cells bound to the endothelium eventually migrate into the cardiac tissue causing significant tissue destruction. Although acute myocardial infarction is not directly caused by inflammation, much of the underlying pathology and the damage that occurs after an acute ischemia-reperfusion injury is caused by acute inflammatory responses during reperfusion, the restoration of blood flow to the affected myocardium. White blood cells present to the area by the newly returning blood release a host of inflammatory factors such as interleukins as well as free radicals in response to tissue damage. The restored blood flow reintroduces oxygen within cells that damages cellular proteins, DNA and the plasma membranes. Damage of the cell membrane may in turn causes release of more free radicals signaling apoptosis. Leukocytes may also build up in small capillaries, obstructing them and leading to more ischemia.
Cardiovascular disease is the leading cause of death in the western world. Coronary artery disease can lead to prolonged or irreversible episodes of cardiac ischemia that result in myocardial infarction (MI) which is associated with a high rate of mortality. The reduced blood flow in heart diseases is typically caused by blockage of a vessel by an embolus (blood clot); the blockage of a vessel due to atherosclerosis; the breakage of a blood vessel (a bleeding stroke); the blockage of a blood vessel due to vasoconstriction such as occurs during vasospasms and possibly, during transient ischemic attacks (TIA) and following subarachnoid haemorrhage. Conditions in which ischemia occurs further include myocardial infarction; trauma; and during cardiac and thoracic surgery and neurosurgery (blood flow needs to be reduced or stopped to achieve the aims of surgery). Procedures that can cause myocardial ischemia include coronary thrombolysis, coronary angioplasty (with or without stent placement), and coronary artery bypass grafts. During myocardial infarct, stoppage of the heart or damage occurs which reduces the flow of blood to myocardium, and ischemia results. Cardiac tissue itself is also subjected to ischemic damage. During various surgeries, reduction of blood flow, clots or air bubbles generated can lead to significant ischemic damage of the myocardium.
During an ischemic event, there is a gradation of injury that arises from the ischemic site. Cells at the site of blood flow restriction, undergo necrosis and form the core of a lesion. A penumbra is formed around the core where the injury is not immediately fatal but progresses slowly toward cell death. This progression to cell death may be reversed upon reestablishment of blood flow within a short time of the ischemic event. Timely reperfusion to reduce the duration of ischemia is the definitive treatment to prevent cellular injury and necrosis in an ischemic myocardium. Typically reperfusion, after a short episode of myocardial ischemia (up to 15 min), is followed by the rapid restoration of cellular metabolism and function. Even with the successful treatment of occluded vessels, a significant risk of additional tissue injury after reperfusion may still occur. If the ischemic episode has been of sufficient severity and/or duration to cause significant changes in the metabolism and the structural integrity of heart muscle, reperfusion may paradoxically result in a worsening of heart function, out of proportion to the amount of dysfunction expected simply as a result of the duration of blocked flow. Although the beneficial effects of early reperfusion of ischemic myocardium with thrombolytic therapy, PTCA, or CABG are now well established, an increasing body of evidence indicates that reperfusion also induces an additional injury to ischemic heart muscle, such as the extension of myocardial necrosis, i.e., extended infarct size and impaired contractile function and metabolism. Hearts undergoing reperfusion after transplantation also undergo similar reperfusion injury events.
Despite efforts towards the development of new therapies for the treatment of diseases and conditions such as heart failure and cardiac ischemia/reperfusion injury, this remains an unmet need for additional or alternative agents to treat or prevent the onset or severity of this condition (Ferdinandy et al., 2007, Pharmacol Rev, 59: 418-458). Current therapies include the use of vasodilators, anti-thrombotics/thrombolytics, beta-blockers and coronary artery bypass graft are used pre and post myocardial ischemia to maintain/restore coronary blood flow and limit oxygen demand.
C. Acute Renal Failure and Related Disease States
Acute renal failure (ARF), also known as acute renal injury (ARI) or acute kidney injury (AKI), is a clinical syndrome characterized by rapid deterioration of renal function that occurs within days. ARF is estimated to occur in at least 5% of all hospitalized patients, and in 30-50% of those admitted to the intensive care unit. ARF secondary to a renal tubular cell injury, including an ischemic injury or a nephrotoxic injury remains a common and potentially devastating problem in clinical medicine and nephrology, with a persistently high rate of mortality and morbidity despite significant advances in supportive care (Chatterjee and Thiemermann, 2003, Expert Opin Emerg Drugs, 8: 389-435).
Pioneering studies over several decades have illuminated the roles of persistent vasoconstriction, tubular obstruction, cellular structural and metabolic alterations, and the inflammatory response in the pathogenesis of ARF. While these studies have suggested possible therapeutic approaches in animal models, translational research efforts in humans have yielded disappointing results. The reasons for this may include the multifaceted response of the kidney to ischemic injury and nephrotoxins, and a paucity of early biomarkers for ARF with a resultant delay in initiating therapy.
The most common cause of ARF—acute tubular necrosis—is most frequently observed in the setting of renal ischemia reperfusion injury, post-renal transplant, sepsis, post-myocardial infarct, in the elderly with diminished fluid intake, and as a consequence of exposure to radiocontrast agents and a wide range of toxins, including cis-platinum, aminoglycosides, amphotericin B, and acyclovir. The notion that only severe renal failure impacts on long-term morbidity is dispelled by the fact that even modest degrees of renal insufficiency significantly increase the risk of death for critically ill patients
Renal ischemia-reperfusion injury refers to an event in which the blood supply to kidneys is obstructed, such as renal occlusion (Chatterjee, 2007, Naunyn-Schmiedeberg's Arch Pharmaco J, 376: 1-43). Reperfusion injury can be defined as the damage that occurs to kidney that is caused by the resumption of blood flow after an episode of ischemia. This damage is distinct from the injury resulting from the ischemia per se. Renal ischemia-reperfusion injury results from several complex and interdependent mechanisms that involve the production of reactive oxygen species, endothelial cell dysfunction, microvascular injury, alterations in intracellular Ca2+ handling, changes in tissue metabolism, and activation of neutrophils, platelets, cytokines and the complement system. All of the deleterious consequences associated with reperfusion constitute a spectrum of reperfusion-associated pathologies that are collectively called reperfusion injury. Reperfusion injury can extend not only acutely, but also over several days following the tissue attack.
Due to obstruction, the endothelial tissue lining the affected blood vessels becomes “sticky” and begins to attract circulating white blood cells (Tohoku, 2008, J Exp Med, 215: 257-266). The white cells bind to the endothelium and eventually migrate into the kidneys causing significant tissue destruction. Although acute renal occlusion is not directly caused by inflammation, much of the underlying pathology and the damage that occurs after an acute ischemia-reperfusion injury is caused by acute inflammatory responses during reperfusion, the restoration of blood flow to the kidney. White blood cells present to the area by the newly returning blood release a host of inflammatory factors such as interleukins as well as free radicals in response to tissue damage. The restored blood flow reintroduces oxygen within cells that damages cellular proteins, DNA, and the plasma membranes. Damage of the cell membrane may in turn promotes release of more free radicals signaling apoptosis. Leukocytes may also built up in small capillaries obstructing them and leading to more ischemia.
During an ischemic event, there is a gradation of injury that arises from the ischemic site. Cells at the site of blood flow restriction, undergo necrosis and form the core of a lesion. A penumbra is formed around the core where the injury is not immediately fatal but progresses slowly toward cell death. This progression to cell death may be reversed upon reestablishment of blood flow within a short time of the ischemic event. Timely reperfusion to reduce the duration of ischemia is the definitive treatment to prevent cellular injury and necrosis in an ischemic kidney. Typically reperfusion, after a short episode of renal ischemia (up to 30 min), is followed by the rapid restoration of cellular metabolism and function. Even with the successful treatment of occluded vessels, a significant risk of additional tissue injury after reperfusion may still occur. If the ischemic episode has been of sufficient severity and/or duration to cause significant changes in the metabolism and the structural integrity of renal tissue, reperfusion may paradoxically result in a worsening of renal function, out of proportion to the amount of dysfunction expected-simply as a result of the duration of blocked flow.
Without being bound by theory acute kidney injury may be the result of renal ischemia-reperfusion injury (ischemia-reperfusion) that occurs, for example, in patients undergoing major surgery such as major cardiac surgery (Padanilam, 2003, Am J Physiol, 284: F608-F627). Renal ischemia, whether caused by shock or during surgery or transplantation, is a major cause of acute kidney injury. Although reperfusion is essential for the survival of ischemic tissue, there is strong evidence that reperfusion itself caused additional cellular injury and together ischemia-reperfusion of the kidney leads to ischemic ARF. Renal ischemia-reperfusion injury is caused by multiple insults involving tubular cell apoptosis, oxygen free radical formation, mitochondrial dysfunction, inflammatory cytokine generation and neutrophils sequestration.
Contrast-induced nephropathy (CIN) is generally recognized as acute renal failure occurring within 48 hours of exposure to intravascular contrast material, and where other causes of renal failure are not attributable. Its presence is generally determined when an increase in serum creatinine levels is exhibited in a subject who has been exposed to intravascular contrast material. CIN is an important problem in clinical practice and contrast-induced morbidity has become a significant cause of hospital morbidity and mortality with the increasing use of iodinated contrast media in diagnostic imaging and interventional procedures such as angiography. In 2003, over 80 million doses of iodinated intravascular contrast media were administrated, corresponding to approximately 8 million liters according to Katzberg et al. (2006, Kidney International, 69: S3-S7). With the increasing use of contrast media in diagnostic and interventional procedures, CIN has become the third leading cause of hospital-acquired acute renal failure, accounting for 10 to 25% of all acute renal failure cases, despite the introduction of newer and safer contrast media, the improvement of hydration protocols, and the introduction of additional preventive strategies.                There are different classes of contrast agents in use such as:        High osmolar agents, such as Iothalamate and Diatrizoate. The osmolality of these agents is about 5 times greater than the osmolality of blood.        Low osmolar agents, such as Iohexyl, Ioversol, Iopamidol, Iopromide, Iomeprol and Ioxaglate. The osmolality of these agents is about 2-3 times greater than the osmolality of blood.        Iso-osmolar agents, such as Iotrolan and Iodixanol. The osmolality of these agents is the same as the osmolality of blood.        
The pathophysiological mechanisms that underlie the development of CIN are not fully understood (ref). Nevertheless, there are recognized risk factors that pre-dispose individuals for the development of contrast agent-induced acute renal failure and these include subject-related factors and procedure-related factors (Kagan and Sheikh-Hamad, 2010, Clinical Cardiology, 33: 62-66). It has been the subject of numerous studies addressing characteristics of the populations at risk and prophylactic strategies. Evidence-based reviews, summarizing recent literature, provides a nephrologists' perspective on contrast-induced nephropathy, focusing on: the pathophysiology of contrast-induced nephropathy; identification of populations at risk; correlation between contrast-induced nephropathy and the type of contrast agent used; and finally, measures to prevent contrast-induced nephropathy, including intravenous fluids, sodium bicarbonate, N-acetylcysteine, and hemofiltration/hemodialysis.
In addition, sepsis and septic shock remain the most important cause of ARF in critically ill patients and account for more than 50% of cases of ARF in the intensive care unit. Despite increasing ability to support vital organs and resuscitate patients, the incidence and mortality of septic ARF remain high. Its mortality varies with the severity of acute kidney injury from 21% to 57%.
Therefore, there is a great need for new, effective strategies for prevention of ARF and in particular therapies for the treatment of diseases and conditions such as I/R injury-, pharmacotherapy-, contrast- and sepsis-induced ARF. This unmet medical need for novel agents to treat, prevent or protect from the onset or severity of these conditions presents opportunities for developing blockbuster drugs.
D. Parstatin: a Protease-Activated Receptor 1-Derived Peptide
Thrombin is a serine protease, which plays a pivotal role in haemostasis. It acts as procoagulant converting fibrinogen into fibrin that anchors platelets at the site of lesion and stabilizes the clot by activating factor XIII and enhances its own generation from prothrombin by activation of factors V, VIII and XI. On the other hand, thrombin acts as an anti-coagulant by activating protein C (Di Cera, 2003).
Apart from its role in blood clotting and fibrin generation, thrombin has important roles in the initiation of angiogenesis (Tsopanoglou and Maragoudakis, 2004, Sem Thromb Hemost, 30: 63-69). Thrombin's angiogenic activity is mostly independent of its coagulant activity and is more dependent on signaling via the protease-activated receptors 1 (PAR1). This supported by the observations obtained in mouse models, wherein a lack of thrombin generation (TF−/−, FX−/−, FV−/−, FII−/−) results in severe vascular defects in embryonic development (Moser and Patterson, 2003, Arterioscler Thromb Vasc Biol, 23: 922-930). Similar phenotypes occur in models of impaired thrombin binding to its PAR receptor (PAR1−/−).
Protease-activated receptors (PARs) consists a family of G protein-coupled receptors which can be activated by proteolytic cleavage of their N-terminal extracellular domain (Ossovskaya and Bunnett, 2004, Physiol Rev, 84: 579-621). PAR1 is the first member of this family to be cloned in which the extracellular amino terminus is cleaved to expose a new amino terminus that is involved in receptor activation (Vu et al., 1991, Cell, 64: 1057-1068). Subsequently, three other members of this receptor family have been identified, designated as PAR2, PAR3 and PAR4. Proteolytic cleavage at the R41/S42 bond of human PAR1 by thrombin releases a 41 amino acid peptide and unveils a tethered peptide ligand with the recognition sequence SFLLRN (SEQ ID NO: 15). This sequence binds to conserved regions in the second extracellular loop of the cleaved receptor, resulting in the initiation of signal transduction. There is evidence that not only thrombin but also other molecules, such as plasmin, factor Xa, activated protein C, as well as matrix metalloprotease-1, might be able to activate this receptor under certain conditions and induce down-stream signals (Leger et al., 2006, Circulation, 114: 1070-1077).
Thrombin, through PAR1 signaling, interacts and stimulates a variety of vascular cells including, but is not limited to, platelets, endothelial cells, smooth muscle cells and regulates the release, expression and activation of the majority of angiogenesis mediators. For example, thrombin-induced angiogenesis in a chick chorioallontoic membrane system is associated with up-regulation of VEGF as well as angiopoietin-2 (Ang-2) (Caunt et al, 2003, J Thromb Haemost, 1: 2097-2102). Also, in endothelial cells thrombin up-regulates VEGF (Huang et al, 2001, Thromb Haemost, 86: 1094-1098), Ang-2 (Huang et al., 2002, Blood, 99: 1646-1650) and the major VEGF receptor KDR (Tsopanoglou and Maragoudakis, 1999, J Biol Chem, 274: 23969-23976), and activates metalloproteinase-2 (Zucher et al., 1995, J Biol Chem, 270: 23730-23738). It was recently shown that thrombin markedly up-regulates growth-regulated oncogene-α and this chemokine in turn mediates the thrombin-induced increase of vascular regulatory proteins (MMP-1, MMP-2), growth factors (VEGF, Ang-2), and receptors (KDR) (Gaunt et al, 2006, Cancer Res, 66: 4125-4132). In addition thrombin induces the secretion of VEGF (Mohle et al., 1997, Proc Natl Acad Sci USA, 94: 663-668) and Ang-1 (Li et al., 2001, Thromb Haemost, 85: 204-206) from platelets. Furthermore, it was demonstrated that thrombin regulates in an opposing fashion the release of VEGF and endostatin (the potent endogenous inhibitor of angiogenesis) in platelets (Ma et al., 2005, Proc Natl Acad Sci USA, 102: 216-220). Thrombin has also been shown to activate the proliferation of endothelial cells by acting directly as mitogenic factor (Olivot et al., 2001, Circ Res, 88: 681-687).
The fact that thrombin plays an important role in angiogenesis, suggests a crucial role for thrombin and its receptor, PAR1 in tumor progression and metastasis (Nierodzik and Karpatkin, 2006, Cancer Cell, 10: 355-362). Thrombin, through PAR1 signaling, contributes to a more malignant phenotype by activating platelet-tumor aggregation, tumor adhesion to subendothelial matrix, tumor growth and metastasis.
In addition, PAR1 expressed on platelets and the vascular endothelium, has been shown to play important roles in normal blood vessel biology (Coughlin, 2005, J Thromb Hemost, 3: 1800-1814) and to contribute to the pathogenesis of several cardiovascular diseases including atherosclerosis, restenosis and thrombosis (Leger et al., 2006, Circulation, 114: 1070-1077). In particular, aberrant over-expression of PAR1 has been documented in the endothelium and vascular smooth muscle cells of human atheroscrerotic arteries, including regions of intimal thickening (Nelken et al., 1992, J Clin Invest, 90: 1614-1621). Activation of PAR1 triggers mitogenic responses in smooth muscle cells and fibroblast and angiogenesis. Targeting PAR1 with a blocking antibody reduced intimal hyperplasia by approximately 50% in a catheter-induced injury model of restenosis (Takada et al., 1998, Circ Res, 82: 980-987). PAR1 deficiency also reduced restenosis in arterial injury models (Cheung et al., 1999, Arterioscler Thromb Vasc Biol, 19: 3014-3024). Recently, it has been shown that thrombin contributes to ischemia/reperfusion injury independently of its effects on platelets and fibrinogen. In addition, PAR1 inhibition has been demonstrated to protect against myocardial ischemia/reperfusion injury by recruiting cardioprotective pathways (Strande et al, 2007, Basic Res Cardiol, 102: 350-358).
Despite the wealth of information relating to the role of thrombin and PAR1 in physiology and diseases states, the information regarding the biological role of cleaved peptides upon activation of PAR1 is very limited. There are only three reports which associate the 41 amino acid cleaved peptide of the PAR1 with potential biological functions (Furman et al., 1998, Proc Natl-Acad Sci USA, 95: 3082-3087; Furman et al., 2000, Thromb Haemost, 84: 897-903; Furman et al., 2003, J Vasc Surg, 37: 440-445). The name of “parstatin” has been coined for this peptide.
The present invention seeks to provide novel peptides derived from parstatin, together with new therapeutic applications for full length parstatin peptide and various peptide fragments thereof.